Enzymes

Experiment

Enzymes

Enzymes

MargaretVorndam,M.S. Version42-0053-00-01

LabReportAssistant

Thisdocumentisnotmeanttobeasubstituteforaformallaboratoryreport.TheLabReportAssistantissimplyasummaryoftheexperiment’squestions,diagramsifneeded,anddatatablesthatshouldbeaddressedinaformallabreport.Theintentistofacilitatestudents’writingoflabreportsbyprovidingthis informationinan editablefilewhichcanbe senttoan instructor.

Observations

Note:Theobservedwellcolorsshouldberecordedaccordingtothefollowingsymbols.Collectdatauntilthewellplateyieldsanambercolor.Then,discontinuetestingthattesttubescontents.

++ = Black or blue-black, Starchpresent

+= LightBlue or Lightblue-black, Some starchpresent

=Amber,Orange,or Red,No starchpresent

Data Table 1: Effect of Temperature on Amylase Enzyme Conversion of Starch to Sugar

Time, minutes

Well/Test Tube 1

0°C

Well/Test Tube 2

37°C

Well/Test Tube 3

Boiling (100°C at STP)

0

++

++

++

5

++

++

++

10

+

+

++

15

+

++

20

+

++

25

++

30

++

Place test tubes that still contain starch in 37°C water bath. Continue timing for 30 minutes.

60

+

Add 1 cm of alpha-amylase solution to test tubes that still contain starch. Place in 37°C water bath. Continue timing for 30 minutes.

90

Data Table 2: The Effect of Concentration on Amylase Enzyme Conversion of Starch to

Sugar

Time in minutes

Test Tube #1

Test Tube

#2

Test Tube

#3

Test Tube

#4

Test Tube

#5

Concentration of

Amylase:

Fill in % per

Test tube

#1 example

0.5 cm ÷ 2 cm, or 1:4

= 25%

16%

12.5%

10%

8.3%

0

++

++

++

++

++

5

++

++

++

++

++

10

+

++

++

++

++

15

+

+

++

++

20

+

+

++

++

25

++

++

30

+

++

If test tube(s) still contains starch, let sit for 30 minutes and retest.

60

Data Table 3: The Effect of pH on Amylase Enzyme Conversion of Starch to Sugar

Time, in minutes

Test Tube #1

Test Tube #2

Test Tube #3

Test Tube #4

pH ( #1 example →)

Student to fill in

pH 3.5

pH 5.0

pH 6.8

pH 11.5

0

++

++

+

+/-

5

++

++

+

10

++

++

+

15

++

++

+/-

20

++

++

25

++

++

30

++

++

If test tube(s) still contains starch, let sit for 30 minutes and retest.

60

Exercise1:EnzymesandTemperature

Questions

  1. Which color indicated that starch was still present in the test tube?

A scale of color indicated the presence of starch in the testtube. The scale of color changes from light blue to black. Light blueindicates that there is a small concentration of starch in the testtube while black indicates that there is a strong concentration ofstarch. In this experiment, the color black and dark blue indicatedthe presence of starch.

  1. Which color indicated that the starch was no longer present?

In the absence of starch,shades of orange and amber were indicated.

  1. When the starch disappeared” as indicated by the color change, what happened to it?

Thestarch disappeared since it had been converted to sugar. When theenzyme reaction was completed it changed to amber.

  1. What is the variable in this exercise?

Thewater temperatures were the variables for this exercise.

Whatcouldbe consideredthe controltreatment?

The amylase and starchsolutions could be considered as the control treatment since theywere used in equal amounts in both test tubes.

D.BasedontheresultsrecordedinDataTable1,whichtemperature(s)is/areoptimalforenzymeconversionactivity?

37°Cis the optimal temperature for enzyme conversion activity.

Which temperaturefacilitatedthe conversionfirst?

The temperature 37° bathfacilitated the first conversion.

Second?

The temperature 0°C bathfacilitated the second conversion.

  1. Did other temperatures facilitate enzyme activity at all?

No,the other temperatures did not facilitate enzyme activity.

Whyor whynot?

Thisis due to the fact that temperatures were not suitable for theenzymes to react. The optimal temperatures needed in order forenzymes to react is 37°C

  1. Why was the water bath temperature of 37°C chosen as the “mid-range” temperature? Hint: What is the oF equivalent of this oC temperature?

This represents theinternal temperature of a normal human being and it was chosen togive the experiment some realty since our bodies work at thistemperature. 98.6°F is the equivalent of 37°C.

  1. Why was it important to immediately observe the color of the test tube contents/iodine in the microplate well?

Observing the color changesimmediately is important due to the fact that the color changes withtime. It is also important to record the data at set intervals so asto be able to determine a timed rate of change. This means that theresults would remain unchanged in case the experiment was repeated.

  1. Why didn’t you add the iodine indicator directly to the test tubes?

Iodine would have taintedthe solution if we had added it directly.

I. Whathappenedwhenyouincubatedthetesttube(s)thatstillcontainedamylase/starchat

37°C?

Theincubated test tubes cooled in less than 30 minutes. The time wasrecorded at the 90°C mark due the laboratory protocol.

Didboth testtubes indicatethe disappearanceof starchaftertheincubation?

Onlyone test tube was incubated.

J. Whathappenedafteryouadded1cmofalpha-amylasesolutiontothetesttube(s)thatstillcontainedstarch,and incubatedthem at37°C?

Adding1 cm of alpha-amylase to the solution diluted the starch furtherallowing the starch to covert to sugar quickly.

Whydid this occur afterthesecondadditionofamylase,but not before?

This is due to the factthat the concentration of amylase was higher which allowed thereactions to take place fast and the temperature was also recorded atbeing optimum.

K. ConstructagraphthatpresentsyourfindingsfromTable1withtimeonthex-axis,andrelativeconcentrationofstarchonthey-axis(++ishighest,+,or–islowest).Includethegraphinthereport.

A.Wasthe initialhypothesissupportedor refuted?Explain.

The test tubes with thehighest concentration of amylase supported my initial hypothesis. Theobservations I made thought the experiment supported my initialhypothesis

B. Whathaveyoulearned fromthis experiment?

The initial concentrationcan be relatively close to the ratio but this does not necessarilymean that this will translate to the time it takes. The test tubeswith the highest concentrations changed first but the change was notequal when a comparison between time and concentration levels wasmade.

C. Whatpracticalapplicationscouldthis knowledgehave?

This experiment would beused practically in laboratories that test enzymes to determine howenzymes affect different substances. This information would also beuseful when manufacturing energy drinks. These drinks should meet theoptimum conditions in order to convert starch in sugar faster.Thereby giving the person taking the drink more energy.

Exercise2: Enzymesand SubstrateConcentration

Questions

  1. What is the variable in this exercise?

The different pH levels arethe variables of this experiment.

B. Whichtesttube has the highestconcentrationofalpha-amylasein the starch?

Testtube one has the highest concentration of alpha amylase at 25%.

  1. How could this experiment be improved?

The experiments that toolong to convert into sugar should have been replaced. This is due tothe fact that it requires a lot of patience yet there is no knowledgethat is gained.

D.ConstructagraphthatpresentsyourfindingsinTable2withtimeonthex-axis,andrelativeconcentrationofstarchonthey-axis(++ishighest,+,or–islowest).Usefivedifferentcolorstorepresentthe concentrationofamylase.Include it in the report.

Discussion

A.Wasyourinitialhypothesissupportedor refutedas towhich testtube starchcontentswouldconverttosugarfirst?

My hypothesis wassupported. The tubes with high concentrations of amylase changed tosugar faster than the tubes with lower concentrations of amylase.This is supported by the observations made.

  1. What have you learned from this experiment?

Ilearned that the initial concentration can be reasonably close inratio but that it does not necessarily translate into the time ittakes. It was observed that higher concentrations saw change firstbut that change was not equal when comparing time and concentrationlevel.

  1. What practical applications could this knowledge have?

A high concentration ofamylase is required to reduce the time needed to convert sugar intostarch.

D.Describeanother waytodo this experimentthatwouldtestthe same concept.

Another alternative waywould be to use equal concentrations of amylase in the test tubes andthen add different amounts of starch to the test tubes. This wouldyield the same results as the concentration which would meet theoptimal conditions would change first.

Exercise3:EnzymesandpH

Questions

A.Whatis the variablein this experiment?

The different pH levelswere the variable in this experiment.

  1. In which test tube(s) did the amylase convert starch to sugar? The tubes with 11.5 and 6.8

  2. Why might you expect this outcome?

This might be expected duethe fact that the higher buffer levels allowed the start to concertto sugar.

C. Didanytesttubes NOTexhibita colorchange?Why?

Both test tube one and twodid not show any change. This is because high starch concentrationand pH levels did not provide the optimal conditions for the enzymesto work. The only optimal conditions that had been met were thetemperature levels and presence of amylase which must be present inorder for the starch to be converted into sugars. The High pH andstarch levels were hence the main reason why the test tubes one andtwo did not exhibit any change.

Discussion

A.Didtheresultssupportorrefuteyourhypothesisastowhichtesttubestarchcontentswouldconverttosugarfirst?

My hypothesis was refuted.I had predicted in my hypothesis that all the test tubes in theexperiment would show some change.

LaboratorySummary

A.Whatother conditionsthatmayaffecttheactionofenzymes?

Availability of substratesto act on is another condition that may affect enzyme action.

B. Howmightyouproposetotestthis?

In order to test for thelevels of concentration, I would lower the concentration of amylaseto the addition tubes 6.8 and 11.

C. Whathaveyoulearned fromthis laboratory?

I have learned the higherthe level of alkalinity the faster the conversation of starch tosugar will take place.

D.Whatpracticalapplicationsmightthis knowledgehave?

A good practicalapplication of this experiment would be in the manufacture of achemical to clean organic wastes. The higher the level of alkalinitythe more the chemical will accelerate the breaking down process.

Copyright© 2008 byMargaretE. Vorndam,1413 CountyRoad671, Rye,CO81069

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